Human Immunity to Malaria The first Kenieroba study (08-I-N120) was completed in 2012 and focused on: 1) Whether Malian children with various hemoglobin and red cell polymorphisms are protected from clinical Plasmodium falciparum malaria; 2) Developing a profile of the acquisition of malaria immunity in Malian children; 3) Examining aspects of pathogenesis due to malaria in Malian children and adults. To initiate these investigations, we conducted a 4-year longitudinal study of 1500 children ranging in age from 6 months to 18 years. All the enrolled children were typed for a series of these red cell and hemoglobin polymorphisms. After diagnosis and treatment of 4207 episodes of malaria, we have developed a very detailed profile of age-dependent acquisition of resistance to malaria in this population. We have shown that sickle cell trait (HbAS) confers greater protection against malaria than other RBC polymorphisms, and a complete analysis was published in 2015. In addition, we continue to analyze a series of samples collected throughout the study. For example, we are testing the ability of sera from these children to limit parasitemias using an antibody-dependent respiratory burst assay. An additional clinical study in Kenieroba was completed in 2015 in conjunction with Dr. Michael Walther, formerly of LMIV (13-I-N209).The primary objective of the study was to demonstrate that co-administration of IV artesunate (AS) and oral activated charcoal (oAC) is not inferior to administration of IV AS alone, with regard to the parasite half-life in children presenting with uncomplicated malaria. To show this we enrolled 35 children into each of two arms, AS + oAC and AS + water. During two transmissions seasons we enrolled 71 children with 70 completing the study. Initial comparative analysis of the two groups showed that there was no significant difference in parasite clearance rate between the two arms indicating that co-administration of IV AS and oAC was not inferior to the administration of IV AS alone. Additionally, there was no difference in the reportable adverse events between the two groups. This data provides the basis for determining whether oAC co-administration with AS can reduce severe malaria in children. Malaria Transmission Transmission of malaria is a critical aspect of the parasite life cycle but is poorly understood, so that we have increased our efforts to investigate parasite sexual stages, malaria transmission in the field, and the biology of the mosquito vector. In May of 2014 we completed a clinical protocol studying 500 individuals of all ages in Kenieroba (13-I-N107). Volunteers were finger-pricked twice per month for a year to analyze both DNA and RNA; in addition, sera were collected by venipuncture three times during the year. This past year we applied the protocols we had previously developed for analysis of parasite DNA by PCR directly on filter paper and we have completed testing of all the samples (>10,000). Using more sensitive molecular methods rather than slide reading, we have found that a relatively high proportion of individuals of all ages carry malaria parasites - in October 2013 (middle of the wet season), P. falciparum prevalence in the cohort was 48.0%, and in March 2014 (middle of the dry season), the prevalence dropped to 18.0%. By a linear regression model, only age and gender showed significant effects on the longitudinal prevalence (p<0.0001 and p=0.0008, respectively), while other host factors did not. In addition, we have established methods for quantitation of gametocyte mRNA encoding Pfs25 using RT-PCR with the RNA samples and we are determining the proportion of parasite positive individuals with gametocytes throughout the year. In addition, selected serum samples are being tested for the capacity to inhibit transmission in membrane feeding assays to determine whether those living in endemic areas develop antibodies that can block transmission and whether they recognize specific sexual stage antigens. Strategies to impact malaria transmission will be accelerated by a better understanding of those individuals primarily responsible for transmission to mosquito vectors. P. falciparum and evasion of mosquito immune system We had obtained experimental evidence that some African strains of P. falciparum can evade the mosquito immune system resulting in more effective transmission and Pfs47 was identified as the malaria gene that allows the parasite to infect mosquitoes without being targeted for destruction. We are testing the hypothesis that the polymorphic Pfs47 gene must interact with a mosquito gene to disrupt the immune response of the vector and that different alleles of Pfs47 work better to evade the immune system of certain mosquito vectors. If this hypothesis is correct, we expect that the different mosquito vectors in Africa will preferentially transmit parasites with certain alleles of Pfs47. The team in Mali has collected mosquitoes from homes using the spray-catch method and performed ELISA assays to identify P. falciparum infected mosquitoes. Material from infected mosquitoes was sent to NIH, genomic DNA was extracted, the Pfs47 gene was sequenced, and the mosquito vectors were typed using molecular tools. We had received a total of 212 mosquito samples sent from Mali and we sequenced the Pfs47 D2 domain (the most variable region) in 150 samples. Two of the 11 haplotypes identified in mosquitoes with a single infection were the most frequent, representing 55 and 18% of the samples; the largest diversity of the 11 haplotypes was found in the month of October. While we did not find a significant difference in the frequency of Pfs47 haplotypes between A. coluzzi and A. gambiae mosquitoes, we are examining other species. An exciting new observation this past year was the finding of naturally occurring Wolbachia infection in Mali. Wolbachia is a bacteria commonly found in various arthropods and is being studied as a vehicle to control insect populations. We are currently obtaining blood-fed females from the field to establish colonies of Wolbachia-infected mosquitoes and study how they interact with the mosquito immune system and Plasmodium falciparum. Dry season ecology of malaria vectors Our main efforts have focused on (1) long distance migration of mosquitoes as a means of re-colonization of Sahelian villages after population extinction during the dry season, and (2) understanding the strategies of persistence of A. coluzzii in the Sahel throughout the dry season. Building on previous results, we have amassed additional compelling evidence that A. coluzzii (previously the M form of A. gambiae) persists locally by aestivation whereas A. gambiae (previously the S form) and A. arabiensis re-colonize the area after the onset of rains. Current major efforts in the field aim to provide direct evidence for these elusive phenomena. Our project on long distance migration combines (i) on-the-ground monitoring of vector density and composition along two transects, over 200 km long, with (ii) aerial sampling of mosquitoes using traps tethered to helium filled balloons. This year, we extended the aerial sample up to 300 m above ground. So far 3 A. gambiae s.l. mosquitoes have been found among other mosquito species. In addition we have established bio-assays to measure flight aptitude of mosquitoes aiming at identifying long-distance migrants on the ground. Both tethered flight assay and free flight assay are currently applied in Mali. Preliminary results show an expected increase in flight activity correlated temporally with presence of A. gambiae in aerial samples. Upon completion of this years fieldwork, we plan to publish the firstreport of wind-assisted long distance migration of malaria vectors. These findings have important implications for malaria transmission and control